title: Activation of RAW264.7 mouse macrophage cells in vitro through treatment with recombinant ricin toxin-binding subunit B: involvement of protein tyrosine, NF-κB and JAK-STAT kinase signaling pathways.
abstract: Ricin toxin-binding subunit B (RTB) is a galactose-binding lectin protein. In the present study, we investigated the effects of RTB on inducible nitric oxide (NO) synthase (iNOS), interleukin (IL)-6 and tumor necrosis factor (TNF)-α, as well as the signal transduction mechanisms involved in recombinant RTB-induced macrophage activation. RAW264.7 macrophages were treated with RTB. The results revealed that the mRNA and protein expression of iNOS was increased in the recombinant RTB-treated macrophages. TNF-α production was observed to peak at 20 h, whereas the production of IL-6 peaked at 24 h. In another set of cultures, the cells were co-incubated with RTB and the tyrosine kinase inhibitor, genistein, the phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, the p42/44 inhibitor, PD98059, the p38 inhibitor, SB203580, the JNK inhibitor, SP600125, the protein kinase C (PKC) inhibitor, staurosporine, the JAK2 inhibitor, tyrphostin (AG490), or the NOS inhibitor, L-NMMA. The recombinant RTB-induced production of NO, TNF-α and IL-6 was inhibited in the macrophages treated with the pharmacological inhibitors genistein, LY294002, staurosporine, AG490, SB203580 and BAY 11-7082, indicating the possible involvement of protein tyrosine kinases, PI3K, PKC, JAK2, p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in the above processes. A phosphoprotein analysis identified tyrosine phosphorylation targets that were uniquely induced by recombinant RTB and inhibited following treatment with genistein; some of these proteins are associated with the downstream cascades of activated JAK-STAT and NF-κB receptors. Our data may help to identify the most important target molecules for the development of novel drug therapies.
authors.affiliation: Department of Immunology, Norman Bethune College of Medical Science, Jilin University, Jilin, P.R. China.
authors.forename: Na
authors.initials: N
authors.lastname: Xu
authors.forename: Hongyan
authors.initials: H
authors.lastname: Yuan
authors.forename: Wensen
authors.initials: W
authors.lastname: Liu
authors.forename: Songyan
authors.initials: S
authors.lastname: Li
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.forename: Jiayu
authors.initials: J
authors.lastname: Wan
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Li
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: Yaping
authors.initials: Y
authors.lastname: Chang
mesh_terms: D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D053612:Janus Kinases / Q000378:metabolism; D008262:Macrophage Activation / Q000187:drug effects* / Q000276:immunology*; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology*; D012276:Ricin / Q000494:pharmacology*; D050791:STAT Transcription Factors / Q000378:metabolism; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: D015850:Interleukin-6; D016328:NF-kappa B; D011994:Recombinant Proteins; D050791:STAT Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D012276:Ricin; D052247:Nitric Oxide Synthase Type II; D011505:Protein-Tyrosine Kinases; D053612:Janus Kinases